Cystic fibrosis transmembrane conductance regulator protein inhibitors and uses thereof

ABSTRACT

The invention provides compositions, pharmaceutical preparations and methods for inhibition of cystic fibrosis transmembrane conductance regulator protein (CFTR) that are useful for the study and treatment of CFTR-mediated diseases and conditions. The compositions and pharmaceutical preparations of the invention may comprise one or more thiazolidinone compounds, and may additionally comprise one or more pharmaceutically acceptable carriers, excipients and/or adjuvants. The methods of the invention comprise, in certain embodiments, administering to a patient suffering from a CFTR-mediated disease or condition, an efficacious amount of a thiazolidinone compound. In other embodiments the invention provides methods of inhibiting CFTR that comprise contacting cells in a subject with an effective amount of a thiazolidinone compound. In addition, the invention features a non-human animal model of CFTR-mediated disease which model is produced by administration of a thiazolidinone compound to a non-human animal in an amount sufficient to inhibit CFTR.

BACKGROUND OF THE INVENTION

[0001] The cystic fibrosis transmembrane conductance regulator protein(CFTR) is a cAMP-activated chloride (Cl⁻) channel expressed inepithelial cells in mammalian airways, intestine, pancreas and testis.CFTR is the chloride-channel responsible for cAMP-mediated Cl−secretion. Hormones, such as a β-adrenergic agonist, or a toxin, such ascholera toxin, leads to an increase in cAMP, activation ofcAMP-dependent protein kinase, and phosphorylation of the CFTR Cl⁻channel, which causes the channel to open. An increase in cell Ca²⁺ canalso activate different apical membrane channels. Phosphorylation byprotein kinase C can either open or shut Cl⁻ channels in the apicalmembrane. CFTR is predominantly located in epithelia where it provides apathway for the movement of Cl− ions across the apical membrane and akey point at which to regulate the rate of transepithelial salt andwater transport. CFTR chloride channel function is associated with awide spectrum of disease, including cystic fibrosis (CF) and with someforms of male infertility, polycystic kidney disease and secretorydiarrhea.

[0002] The hereditary lethal disease cystic fibrosis (CF) is caused bymutations in CFTR. Observations in human cystic fibrosis (CF) patientsand CF mouse models indicate the functional importance of CFTR inintestinal and pancreatic fluid transport, as well as in male fertility(Grubb et al., 1999, Physiol. Rev. 79:S193-S214; Wong, P. Y., 1997, Mol.Hum. Reprod. 4:107-110). However, the mechanisms remain unclear by whichdefective CFTR produces airway disease, which is the principal cause ofmorbidity and mortality in CF (Pilewski et al., 1999, Physiol. Rev.79:S215-S255.). Major difficulties in understanding airway disease in CFinclude the inadequacy of CF mouse models, which manifest little or noairway disease, the lack of large animal models of CF, and the limitedavailability of human CF airways that have not been damaged by chronicinfection and inflammation. High-affinity, CFTR-selective inhibitorshave not been available to study airway disease mechanisms in CF or tocreate the CF phenotype in large animal models.

[0003] High-affinity CFTR inhibitors also have clinical applications inthe therapy of secretary diarrheas and cystic kidney disease, and ininhibiting male fertility. The compounds diphenylamine-2-carboxylate(DPC) and 5-nitro-2(3-phenylpropyl-amino)benzoate (NPPB) inhibit CFTR athigh concentrations but are non-specific in their inhibitory action(Cabantchik et al., 1992, Am. J Physiol. 262:C803-C827; McDonough etal., 1994, Neuron 13:623-634; Schultz et al., 1999, Physiol. Rev.79:S109-S144.). The best CFTR inhibitor available forelectrophysiological and other cell-based studies, glibenclamide, isused at concentrations of >100 μM (Sheppard et al., 1992, J. Gen.Physiol. 100:573-591; Hongre et al, 1994, Pfugers Arch. 426:284-287).However, at this concentration glibenclamide also inhibits other Cl⁻transporters as well as K⁺ channels (Edwards et al., 1993, Br. J.Pharmacol. 110:1280-1281; Rabe et al., 1995, Pflugers Arch. 429:659-662;Yamazaki et al., 1997, Circ. Res. 81:101-109). Effective small moleculeinhibitors of other ion transport proteins are known, but no smallmolecules with specific CFTR inhibitory ability suitable for therapy ofsecretory diseases have been available.

[0004] There is accordingly a need in for CTFR inhibitor compounds andmethods of using such compounds for development of animal models usefulin the study and treatment of CF and the treatment and control ofsecretory disorders. The present invention addresses these needs, aswell as others, and overcomes deficiencies found in the background art.

SUMMARY OF THE INVENTION

[0005] The invention provides compositions, pharmaceutical preparationsand methods for inhibition of cystic fibrosis transmembrane conductanceregulator protein (CFTR) that are useful for the study and treatment ofCFTR-mediated diseases and conditions. The compositions andpharmaceutical preparations of the invention may comprise one or morethiazolidinone compounds, and may additionally comprise one or morepharmaceutically acceptable carriers, excipients and/or adjuvants. Themethods of the invention comprise, in certain embodiments, administeringto a patient suffering from a CFTR-mediated disease or condition, anefficacious amount of a thiazolidinone compound. In other embodimentsthe invention provides methods of inhibiting CFTR that comprisecontacting cells in a subject with an effective amount of athiazolidinone compound. In addition, the invention features a non-humananimal model of CFTR-mediated disease which model is produced byadministration of a thiazolidinone compound to a non-human animal in anamount sufficient to inhibit CFTR.

[0006] These and other objects and advantages of the invention will beapparent from the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The invention will be more fully understood by reference to thefollowing drawings, which are for illustrative purposes only.

[0008]FIG. 1A is a schematic representation of a screening techniqueused for detection of CFTR inhibitors. CFTR was maximally stimulated bymultiple agonists in stably transfected epithelial cells co-expressinghuman CFTR and a yellow fluorescent protein (YFP) having Cl⁻/I⁻sensitive fluorescence. After addition of a test compound, I⁻ influx wasinduced by adding an I⁻ containing solution.

[0009]FIG. 1B is a graphical illustration of representative fluorescencedata from individual wells using the screening technique of FIG. 1A,showing controls (no activator, no test compound), inactive compoundsand active CFTR inhibitor compounds.

[0010]FIG. 1C shows chemical structures of 2-thioxo-4-thiazolidinoneCFTR inhibitors identified by the screening technique of FIG. 1A.

[0011]FIG. 1D shows chemical structures of Ring 2 of the2-thioxo-4-thiazolidinone analogs having the greatest CFTR inhibitoryactivity. The complete 2-thioxo-4-thiazolidinone structure is shown inFIG. 1C. Relative potencies were: 0.2 (CFTR_(inh)-020), 0.3(CFTR_(inh)-029), 1.0 (CFTR_(inh)-172), 0.2 (CFTR_(inh)-185), 0.1(CFTR_(inh)-214) and 0.1 (CFTR_(inh)-236).

[0012]FIG. 2A is a graphical representation of relative fluorescenceversus time using the screening technique of FIG. 1A for the CFTRinhibitor3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(referred to herein as CFTR_(inh)-172) at several concentrations.

[0013]FIG. 2B is a graphical representation of the time course ofinhibition showing CFTR-mediated I- transport rates at different timesafter addition of 2 μM CFTR_(inh)-172. The inset is a graphicalrepresentation of the time course of inhibition reversal showing I⁻transport rates at different times after washout of 1 μM CFTR_(inh)-172.Mean ± SE from three sets of experiments.

[0014]FIG. 2C is a graphical representation of inhibition of CFTR afterstimulation by different agonists, including benzoflavone andbenzimidazolone UCCF compounds (UC_(CF)-029(2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate) and UC_(CF)-853(Galietta et al. 2001 J. Biol. Chem. 276:19723-19728), genistein,CPT-cAMP, 8-methoxypsoralen (8-MPO), 8-cyclopentyl-1,3-dipropylxanthine(CPX) (all 50 μM) (±SE from three sets of experiments). Filled bars showagonist, and open bars show agonist with 5 μM CFTR_(inh)-172.

[0015]FIG. 3A is a graphical representation of CFTR_(inh)-172 inhibitionof short-circuit current in permeabilized FRT cells expressing humanCFTR. CFTR was stimulated by 100 μM CPT-cAMP.

[0016]FIG. 3B graphically provides a summary of dose-inhibition data forCFTR_(inh)-172 (circles) and glibenclamide (squares) (SE, three sets ofexperiments).

[0017]FIG. 3C graphically illustrates CFTR_(inh)-172 inhibition ofshort-circuit current in primary culture of (non-permeabilized) humanbronchial epithelial cells. Inhibitor was added in apical bathingsolution (left panel) or basolateral and then apical solutions (rightpanel).

[0018]FIG. 3D is a graphical representation of whole-cell patch clamp ofCFTR-expressing FRT cells showing membrane currents elicited at +80 mV(open circles) and −100 mV (closed circles). CFTR was stimulated by 5 μMforskolin followed by addition of 2 μM CFTR_(inh)-172.

[0019]FIG. 3E is a graphic illustration showing that alternatestimulation was interrupted (a-c) to apply graded membrane potentials.

[0020]FIG. 3F is a graphical representation of current-voltagerelationships under basal conditions (control, open circles), afterforskolin stimulation (filled circles), and following addition of 0.2 μMCFTR_(inh)-172 giving ˜50% inhibition (open triangles).

[0021]FIG. 4A is a graphical representation of UTP- (100 μM) stimulatedCa²⁺-dependent Cl⁻ secretion measured in short-circuit currentmeasurements on airway epithelial cells in the absence and presence of 5μM of CFTR_(inh)-172.

[0022]FIG. 4B is a graphical representation of volume-activated Cl⁻current (hypotonic 250 mosM/kg H₂O) measured in whole-cell patch clampexperiments on FRT cells. Currents were recorded in the absence andpresence of 5 μM CFTR_(inh)-172.

[0023]FIG. 4C is a graphical representation of ³H-vincristineaccumulation in 9HTEo-/Dx cells with upregulated MDR-1expression.Intracellular vincristine was measured with and without verapamil (100μM) or CFTR_(inh)-172 (5 μM) (SE, n=3).

[0024]FIG. 4D is a graphical illustration showing a representativemembrane potential recording from a pancreatic β cell (INS-1) perfusedextracellularly with CFTR_(inh)-172, diazoxide (100 μM), andglibenclamide (10 μM).

[0025]FIG. 4E is a graphical representation of averaged changes inmembrane potential (ΔmV) caused by maneuvers indicated in FIG. 4D (SE,n=4). FIG. 5A is a photograph of isolated mouse ileal loops at six hoursafter lumenal injection of 1 μg cholera toxin without (top) and with(middle) intraperitoneal injection of CFTR_(inh)-172 (150 μg/kg). Asaline control (no cholera toxin, bottom) is shown for comparison.

[0026]FIG. 5A is a photograph of isolated mouse ileal loops at six hoursafter lumenal injection of 1 μg cholera toxin without (top) and with(middle) intraperitoneal injection of CFTR_(inh)-172 (150 μg/kg). Asaline control (no cholera toxin, bottom) is shown for comparison.

[0027]FIG. 5B graphically illustrates ileal loop weight at six hours,with a mean ± SE (n=6-8 mice) with 14-16 loops studied. For the inactiveanalog, the 4-carboxyphenyl group in CFTR_(inh)-172 was replaced by3-methoxy-4-methoxyvinylphenyl (SE, 6-8 mice per group, * p<0.001,ANOVA).

[0028]FIG. 5C graphically illustrates the ratio of weight of entiresmall intestine at six hours after oral gavage before vs. after luminalfluid removal (SE, 4 mice per group, p<0.001).

[0029]FIG. 5D is a graphical illustration showing a representativeCFTR_(inh)-172 inhibition short-circuit current after amiloride additionand stimulation by forskolin (20 μM) in isolated rat colonic mucosa.CFTR_(inh)-172 added to serosal and then mucosal surfaces as indicated(n=4).

[0030] Before the present invention is described, it is to be understoodthat this invention is not limited to particular embodiments described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

[0031] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0032] It should be noted that, as used herein and in the appendedclaims, the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an inhibitor” includes a plurality of suchinhibitors, and reference to “the cell” includes reference to one ormore cells and equivalents thereof known to those skilled in the art,and so forth.

[0033] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application, and areincorporated herein by reference. Nothing herein is to be construed asan admission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication datesthat may need to be independently confirmed.

[0034] The definitions used herein are provided for reason of clarity,and should not be considered as limiting. The technical and scientificterms used herein are intended to have the same meaning as commonlyunderstood by those of ordinary skill in the art to which the inventionpertains.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention is based on the discovery of thiazolidinonecompounds that are high-affinity CFTR inhibitors. The structure of thecompounds of the invention, as well as pharmaceutical formulations andmethods of use are described in more detail below.

[0036] Definitions

[0037] A “cystic fibrosis transmembrane conductance regulatorprotein-mediated condition or symptom” or “CFTR-mediated condition orsymptom” means any condition, disorder or disease, or symptom of suchcondition, disorder, or disease, that results from activity of cysticfibrosis transmembrane conductance regulator protein (CFTR), e.g.,activity of CFTR in ion transport. Such conditions, disorders, diseases,or symptoms thereof are treatable by inhibition of CFTR activity, e.g.,inhibition of CFTR ion transport. CFTR activity has been implicated in,for example, intestinal secretion in response to various agonists,including cholera toxin (see, e.g., Snyder et al. 1982 Bull. WorldHealth Organ. 60:605-613; Chao et al. 1994 EMBO J. 13:1065-1072; Kimberget al. 1971 J. Clin. Invest.50:1218-1230).

[0038] A “CFTR inhibitor” as used herein is a compound that reduces theefficiency of ion transport by CFTR, particularly with respect totransport of chloride ions by CFTR. Preferably CFTR inhibitors of theinvention are specific CFTR inhibitors, i.e., compounds that inhibitCFTR activity without significantly or adversely affecting activity ofother ion transporters, e.g., other chloride transporters, potassiumtransporters, and the like. Preferably the CFTR inhibitors arehigh-affinity CFTR inhibitors, e.g., have an affinity for CFTR of atleast about one micromolar, usually about one to five micromolar.

[0039] “Treating” or “treatment” of a condition or disease includes: (1)preventing the disease, i.e. causing the clinical symptoms of thedisease not to develop in a mammal that may be exposed to or predisposedto the disease but does not yet experience or display symptoms of thedisease, (2) inhibiting the disease, i.e., arresting or reducing thedevelopment of the disease or its clinical symptoms, or (3) relievingthe disease, i.e., causing regression of the disease or its clinicalsymptoms.

[0040] A “therapeutically effective amount” or “efficacious amount”means the amount of a compound that, when administered to a mammal orother subject for treating a disease, is sufficient to effect suchtreatment for the disease. The “therapeutically effective amount” willvary depending on the compound, the disease and its severity and theage, weight, etc., of the subject to be treated.

[0041] The terms “subject” and “patient” mean a member or members of anymammalian or non-mammalian species that may have a need for thepharmaceutical methods, compositions and treatments described herein.Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), avian, and other subjects. Humans and non-humananimals having commercial importance (e.g., livestock and domesticatedanimals) are of particular interest.

[0042] “Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primat, murine, lagomorpha, etc. maybe used for experimental investigations.

[0043] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular terpeneor terpenoid compound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

[0044] The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

[0045] A “pharmaceutically acceptable excipient” means an excipient thatis useful in preparing a pharmaceutical composition that is generallysafe, non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. “A pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

[0046] A “pharmaceutically acceptable salt” of a compound means a saltthat is pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4methylbicyclo>2.2.2!oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

[0047] “Pro-drugs” means any compound that releases an active parentdrug according to formula (I) in vivo when such prodrug is administeredto a mammalian subject. Prodrugs of a compound of formula (I) areprepared by modifying functional groups present in the compound offormula (I) in such a way that the modifications may be cleaved in vivoto release the parent compound. Prodrugs include compounds of formula(I) wherein a hydroxy, amino, or sulfhydryl group in compound (I) isbonded to any group that may be cleaved in vivo to regenerate the freehydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugsinclude, but are not limited to esters (e.g., acetate, formate, andbenzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) ofhydroxy functional groups in compounds of formula (I), and the like.

[0048] The term “organic group” and “organic radical” as used hereinmeans any carbon-containing group, including hydrocarbon groups that areclassified as an aliphatic group, cyclic group, aromatic group,functionalized derivatives thereof and/or various combination thereof.The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈ ) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

[0049] “Organic groups” may be functionalized or otherwise compriseadditional functionalities associated with the organic group, such ascarboxyl, amino, hydroxyl, and the like, which may be protected orunprotected. For example, the phrase “alkyl group” is intended toinclude not only pure open chain saturated hydrocarbon alkylsubstituents, such as methyl, ethyl, propyl, t-butyl, and the like, butalso alkyl substituents bearing further substituents known in the art,such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro,amino, carboxyl, etc. Thus, “alkyl group” includes ethers, esters,haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

[0050] The terms “halo” and “halogen” refer to the fluoro, chloro, bromoor iodo groups. There can be one or more halogen, which are the same ordifferent. Preferred halogens are chloro and fluoro.

[0051] The term “cycloalkyl” means a mono-, bi-, or tricyclic saturatedring that is fully saturated or partially unsaturated. Examples of sucha group included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

[0052] The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

[0053] The term “substituted phenyl” specifies a phenyl groupsubstituted with one or more moieties, and in some instances one, two,or three moieties, chosen from the groups consisting of halogen,hydroxy, protected hydroxy, cyano, nitro, trifluoromethyl, C1 to C7alkyl, C1 to C7 alkoxy, C1 to C7 acyl, C1 to C7 acyloxy, carboxy,oxycarboxy, protected carboxy, carboxymethyl, protected carboxymethyl,hydroxymethyl, protected hydroxymethyl, amino, protected amino,(monosubstituted)amino, protected (monosubstituted)amino,(disubstituted)amino, carboxamide, protected carboxamide, N-(C1 to C6alkyl)carboxamide, protected N-(C1 to C6 alkyl)carboxamide, N,N-di(C1 toC6 alkyl)carboxamide, trifluoromethyl, N-((C1 to C6alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl, substituted orunsubstituted, such that, for example, a biphenyl or naphthyl groupresults.

[0054] Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

[0055] The term “(substituted phenyl)alkyl” means one of the abovesubstituted phenyl groups attached to one of the above-described alkylgroups. Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

[0056] As noted above, the term “aromatic” or “aryl” refers to five andsix membered carbocyclic rings. Also as noted above, the term“heteroaryl” denotes optionally substituted five-membered orsix-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfurand/or nitrogen atoms, in particular nitrogen, either alone or inconjunction with sulfur or oxygen ring atoms. These five-membered orsix-membered rings may be fully unsaturated.

[0057] Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to a aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, and preferably to a benzene ring.

[0058] The following ring systems are examples of the heterocyclic(whether substituted or unsubstituted) radicals denoted by the term“heteroaryl”: thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl,isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl,thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl,oxazinyl, triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl andpurinyl, as well as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

[0059] Substituents for the above optionally substituted heteroarylrings are from one to three halo, trihalomethyl, amino, protected amino,amino salts, mono-substituted amino, di-substituted amino, carboxy,protected carboxy, carboxylate salts, hydroxy, protected hydroxy, saltsof a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl,substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl,and (substituted phenyl)alkyl. Substituents for the heteroaryl group areas heretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C1 to C4 alkoxy group, similarly, “loweralkylthio” means a C1 to C4 alkylthio group.

[0060] The term “(monosubstituted)amino” refers to an amino group withone substituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C1 to C4 acyl, C2 to C7 alkenyl, C2 toC7 substituted alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1to C7 acyl, C2 to C7 alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7to C16 substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

[0061] The term “heteroaryl(alkyl)” denotes an alkyl group as definedabove, substituted at any position by a heteroaryl group, as abovedefined.

[0062] “Optional” or “optionally” means that the subsequently describedevent, circumstance, feature or element may, but need not, occur, andthat the description includes instances where the event or circumstanceoccurs and instances in which it does not. For example, “heterocyclogroup optionally mono- or di- substituted with an alkyl group” meansthat the alkyl may, but need not, be present, and the descriptionincludes situations where the heterocyclo group is mono- ordisubstituted with an alkyl group and situations where the heterocyclogroup is not substituted with the alkyl group.

[0063] Compounds that have the same molecular formula but differ in thenature or sequence of bonding of their atoms or the arrangement of theiratoms in space are termed “isomers.” Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers.”Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers.” When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture.”

[0064] The compounds of this invention may possess one or moreasymmetric centers; such compounds can therefore be produced asindividual (R)- or (S)-stereoisomers or as mixtures thereof. Unlessindicated otherwise, the description or naming of a particular compoundin the specification and claims is intended to include both individualenantiomers and mixtures, racemic or otherwise, thereof. The methods forthe determination of stereochemistry and the separation of stereoisomersare well-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

[0065] Overview

[0066] The invention provides thiazolidinone compositions and methods oftheir use in high affinity inhibition of cystic fibrosis transmembraneconductance regulator protein (CFTR) and for the study and treatment ofCFTR-mediated diseases and conditions. The discovery of the subjectthiazolidinone compounds was based on screening of numerous potentialcandidate compounds using an assay designed to identify CFTR inhibitorsthat interact directly with CFTR. Without being held to any particulartheory or mode of operation, since multiple CFTR activators that work ondifferent activating pathways were included in the studies leading toidentification of the subject compounds, the inhibitory compounds of theinvention likely effect inhibition by acting at or near the CFTR Cl⁻transporting pathway. A screening of 50,000 diverse compounds identifiedseveral 2-thioxo-4-thiazolidinone compounds as effective CFTRinhibitors. These compounds are unrelated chemically and structurally topreviously known CFTR activators or to the previously known CFTRinhibitors DPC, NPPB or glibenclamide. The most potent CFTR inhibitoridentified from screening had a K₁ of ˜300 nM for inhibition of Cl⁻current in human airway cells. Inhibition was rapid, reversible andCFTR-specific.

[0067] The compositions and methods of the invention will now bedescribed in more detail.

[0068] Thiazolidinone Compounds

[0069] The thiazolidinone compounds used in the compositions and methodsof the invention comprise a heterocyclic ring of five or more atoms,including an aryl substituted nitrogen, at least one sulfur, oxygen orselenium heteroatom, and one or more carbonyl or thiocarbonyl groupsassociated with the heterocyclic ring. More specifically, the subjectthiazolidinone compounds may comprise the formula

[0070] wherein X₁, X₂ and X₃ each individually are hydrogen, any organicgroup, any halo group, or a nitro group, azo group, hydroxyl group orthio group, Y₁, Y₂ and Y₃ each individually comprise hydrogen, anyorganic group, any halo group, or a nitro group, azo group, hydroxylgroup or thio group, A₁ and A₂ each individually are oxygen or sulfur,A₃ is sulfur or selenium, and A₄ comprises one or more carbons orheteroatoms and may be present or absent. Where A₄ is absent the centralheterocyclic ring is a five membered ring.

[0071] In certain embodiments, the thiazolidinone compounds comprise theformula:

[0072] wherein X is hydrogen, any organic group, any halo group, or anitro group, azo group, hydroxyl group or thio group, Y₁, Y₂ and Y₃ eachindividually are hydrogen, any organic group, any halo group, or a nitrogroup, azo group, hydroxyl group or thio group, and A₁ and A₂ eachindependently are oxygen or sulfur. In specific embodiments, X may be anelectron withdrawing group, and may comprise a haloalkyl group,dihaloalkyl group, trihaloalkyl group (e.g., trifluoroalkyl group) or afluoro group. Y₁ may be selected from the group consisting of alkyl,hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl,and halo groups, Y₂ may be selected from the group consisting ofhydroxyl and bromo groups, and Y₃ may be selected from the groupconsisting of hydrogen and a nitro group.

[0073] The subject thiazolidinone compounds in many embodiments maycomprise 3-aryl-5-arylmethylene-2-thioxo-4-thiazolidinones of theformula

[0074] wherein X is any electronegative or electron withdrawing group,and Y₁, Y₂ and Y₃ each individually are hydrogen, alkyl, hydroxyl,carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, or halogroups. In one embodiment X is at position selected from 2, 3, or 4; Y₁,is at a position selected from 2, 3, or 4; and Y₂ and Y₃ may behydrogen. The 3-aryl-5-arylmethylene-2-thioxo-4-thiazolidinones may morespecifically have the formula

[0075] wherein Y₁-Y₃ are as described above. In one embodimenttrifluromethyl group is at a position selected from 2, 3, or 4; Y₁ is ata position selected from 2, 3, or 4; where Y₂ and Y₃ may be hydrogen inthis embodiment.

[0076] In some embodiments of the invention, the thiazolidinonecompounds may comprise:

[0077]3-[(3-trifluoromethyl)phenyl]-5-[(4-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone;

[0078]3-[(3-trifluoromethyl)phenyl]-5-[(4-oxycarboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;

[0079]3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;

[0080]3-[(3-trifluoromethyl)phenyl]-5-[(3,4-dihydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone;

[0081]3-[(3-trifluoromethyl)phenyl]-5-[(3,5-dibromo-4-hydroxyphenyl)methylenel-2-thioxo-4-thiazolidinone;and

[0082]3-[(3-trifluoromethyl)phenyl]-5-[(3-bromo-4-hydroxy-5-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone.The trifluromethyl group may be at positions 2, 3, or 4 in any of theabove-recited compounds.

[0083] Pharmaceutical Preparations

[0084] Also provided by the invention are pharmaceutical preparations ofthe subject thiazolidinone compounds described above. The subjectcompounds can be incorporated into a variety of formulations fortherapeutic administration by a variety of routes. More particularly,the compounds of the present invention can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers, diluents, excipients and/oradjuvants, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.Preferably, the formulations are free of detectable DMSO (dimethylsulfoxide), which is not a pharmaceutically acceptable carrier, diluent,excipient, or adjuvant. The formulations may be designed foradministration to subjects or patients in need thereof via a number ofdifferent routes, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration.

[0085] In pharmaceutical dosage forms, the subject compounds of theinvention may be administered in the form of their pharmaceuticallyacceptable salts, or they may also be used alone or in appropriateassociation, as well as in combination, with other pharmaceuticallyactive compounds. The following methods and excipients are merelyexemplary and are in no way limiting.

[0086] For oral preparations, the subject compounds can be used alone orin combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0087] The subject compounds of the invention can be formulated intopreparations for injection by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives.

[0088] The compounds of the invention can be utilized in aerosolformulation to be administered via inhalation. The compounds of thepresent invention can be formulated into pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen and thelike.

[0089] Furthermore, the subject compounds can be made into suppositoriesby mixing with a variety of bases such as emulsifying bases orwater-soluble bases. The compounds of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

[0090] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

[0091] Depending on the subject and condition being treated and on theadministration route, the subject compounds may be administered indosages of, for example, 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.The inventors have found that cholera toxin-induced intestinal fluidsecretion in mice is effectively blocked by a single intraperitonealdose of about 10-20 micrograms with a dosage of about ten times greaterbeing effective in rats. Thus, for example, oral dosages may be aboutten times the injection dose. Higher doses may be used for localizedroutes of delivery.

[0092] A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

[0093] For use in the subject methods, the subject compounds may beformulated with other pharmaceutically active agents, including otherCFTR-inhibiting agents.

[0094] Pharmaceutically acceptable excipients usable with the invention,such as vehicles, adjuvants, carriers or diluents, are readily availableto the public. Moreover, pharmaceutically acceptable auxiliarysubstances, such as pH adjusting and buffering agents, tonicityadjusting agents, stabilizers, wetting agents and the like, are readilyavailable to the public.

[0095] Those of skill in the art will readily appreciate that doselevels can vary as a function of the specific compound, the severity ofthe symptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

[0096] Kits with unit doses of the subject compounds, usually in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the drugs intreating pathological condition of interest. Preferred compounds andunit doses are those described herein above.

[0097] Conditions Amenable to Treatment Using the CFTR Inhibitors of theInvention

[0098] The CFTR inhibitors disclosed herein are useful in the treatmentof a CFTR-mediated condition, i.e., any condition, disorder or disease,or symptom of such condition, disorder, or disease, that results fromactivity of CFTR, e.g., activity of CFTR in ion transport. Suchconditions, disorders, diseases, or symptoms thereof are amenable totreatment by inhibition of CFTR activity, e.g., inhibition of CFTR iontransport.

[0099] In one embodiment, the CFTR inhibitors of the invention are usedin the treatment of conditions associated with aberrantly increasedintestinal secretion, particularly acute aberrantly increased intestinalsecretion. CFTR activity has been implicated in intestinal secretion inresponse to various agonists, including cholera toxin (see, e.g., Snyderet al. 1982 Bull. World Health Organ. 60:605-613; Chao et al. 1994 EMBOJ. 13:1065-1072; Kimberg et al. 1971 J Clin. Invest.50:1218-1230). ThusCFTR inhibitors of the invention can be administered in an amounteffective to inhibit CFTR ion transport and thus decrease intestinalfluid secretion.

[0100] Thus, CFTR inhibitors can be used in the treatment of intestinalinflammatory disorders and diarrhea, particularly secretory diarrhea.Secretory diarrhea is the biggest cause of infant death in developingcountries, with about 5 million deaths annually (Gabriel et al., 1994Science 266: 107-109). Several studies, including those using CF mice,indicate that CFTR is the final common pathway for intestinal chlorideion (and thus fluid) secretion in response to various agonists (Snyderet al., 1982, Bull. World Health Organ. 60: 605-613; Chao et al., 1994EMBO. J. 13: 1065-1072; and Kimberg et al., 1971, J. Clin. Invest. 50:1218-1230.). The mouse models of intestinal fluid secretion used hereinindicate that CFTR inhibition by systemic administration of theinhibitor at a non-toxic dose effectively blocked intestinal fluidsecretion induced by cholera toxin (see Examples).

[0101] Diarrhea can result from exposure to a variety of pathogens oragents including, without limitation, cholera toxin (Vibrio cholera),enterotoxigenic E. coli (ETEC), food poisoning, or other toxin exposurethat results in increased intestinal secretion mediated by CFTR.

[0102] CFTR inhibitors may also be useful in the treatment of diarrheaassociated with AIDS (e.g., AIDS-related diarrhea), and inflammatorygastrointestinal disorders, such as ulcerative colitis, inflammatorybowel disease (IBD), Crohn's disease, and the like. It has been reportedthat intestinal inflammation modulates the expression of three majormediators of intestinal salt transport and may contribute to diarrhea inulcerative colitis both by increasing transepithelial Cl− secretion andby inhibiting the epithelial NaCl absorption (see, e.g., Lohi et al. AmJ Physiol Gastrointest Liver Physiol 2002 Sep;283(3):G567-75).

[0103] CFTR inhibitors of the invention can also be used in treatment ofconditions such as polycystic kidney disease, and find further use asmale infertility drugs, by inhibition of CFTR activity in the testis.

[0104] CFTR inhibitors of the invention can be further screened inlarger animal models (e.g., the rabbit model described in Spira et al.,1981, Infect. Immun. 32:739-747.). In addition, analysis of stool outputusing live Vibrio cholerae can also be examined to further characterizethe CFTR inhibitors of the invention.

[0105] Non-Human Animal Models and Human Tissue Models ofCFTR-Deficiencies

[0106] The CFTR inhibitors of the invention can also be used to generatenon-human animal models of disease, where the disease is associated withdecreased CFTR function (e.g., decreased ion transport). There isincreasing evidence that defective fluid and macromolecular secretion byairway submucosal glands leads to impaired mucociliary and bacterialclearance in CFTR-deficient subjects, particularly in those affectedwith cystic fibrosis (CF); however, functional studies in human airwayglands have been restricted to severely diseased airways obtained at thetime of lung transplantation (Jayaraman et al. 2001 Proc. Natl. Acad.Sci. USA 98:8119-8123). Acute CFTR inhibition permits determination ofthe role of CFTR in water, salt and macromolecule secretion bysubmucosal glands. High-affinity CFTR inhibitors permit thepharmacological creation of non-human animal models that mimicCFTR-deficiency in humans, e.g., mimics the human CF phenotype. Inparticular, large animal models of CFTR deficiency (e.g., CF) findparticular use in elucidating the pathophysiology of initiation andprogression of airway disease in CF, and in evaluating the efficacy ofCF therapies, e.g., screening candidate agents for treatment ofCFTR-deficiencies or symptoms thereof.

[0107] Inhibition of CFTR ion transport can be manifested in airway andpancreatic disorders, as well as infertility in males. For example,inhibition of CFTR channels in the lungs and airways influences airwaysurface fluids leading to accumulation of mucus, which in turn plugsairways and collects heavily on the lung walls, providing a primeenvironment for infection to occur, which in turn can lead to chroniclung disease. This same phenomenon occurs in the pancreas, where theaccumulated mucus disrupts the exocrine function of the pancreas andprevents essential food-processing enzymes from reaching the intestines.

[0108] Such non-human animal models can be generated by administrationof an amount of a CFTR inhibitor effective to decrease CFTR activity inion transport. Of particular interest is the use of the CFTR inhibitorsof the invention to induce the cystic fibrosis (CF) phenotype in anon-human animal. Administration of an amount of a CFTR inhibitoreffective to inhibit CFTR receptors in, for example, lung effectivelymimics the CFTR defect found in CF. Routes of delivery for CFTRinhibitor are discussed in detail above. Depending on the non-humananimal used, the subject compounds may be administered in dosages of,for example, 50 to 500 μg/kg body weight one to three times a day by anintraperitoneal, subcutaneous, or other route to generate the non-humananimal models. Oral dosages may be up to about ten times theintraperitoneal or subcutaneous dose.

[0109] Non-human animal models of CFTR-associated disease can be used asmodels of any appropriate condition associated with decreased CFTRactivity. Such conditions include those that are associated with CFTRmutations, which mutations result in abnormalities in epithelial ion andwater transport. These abnormalities can in turn be associated withderangements in airway mucociliary clearance, as well as in othermucosal epithelia and ductal epithelia. Conditions that can bepharmacologically modeled by inducing a CFTR-deficient phenotype in anon-human animal include, without limitation, cystic fibrosis (includingatypical CF), idiopathic chronic pancreatitis, vas deferens defects,mild pulmonary disease, asthma, and the like. For a review of disordersassociated with impaired CFTR function, see, e.g., Noone et al. RespirRes 2 328-332 (2001). CFTR inhibitor-generated non-human animal modelscan also serve as models of microbial infection (e.g., bacterial, viral,or fungal infection, particularly respiratory infections) in aCFTR-deficient subject. In one embodiment of particular interest, theCFTR inhibitors of the invention are used to pharmacologically inducethe cystic fibrosis (CF) phenotype.

[0110] Animals suitable for use in the production of the animal modelsof the invention include any animal, particularly a mammal, e.g.,non-human primates (e.g., monkey, chimpanzee, gorilla, and the like),rodents (e.g., rats, mice, gerbils, hamsters, ferrets, and the like),lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline,and the like. Large animals are of particular interest.

[0111] The CFTR inhibitors can also be contacted with isolated humantissue to create ex vivo models of disease. Such tissue is contactedwith an amount of a CFTR inhibitor effective to decrease CFTR activityin the tissue, which may be for as little as 15 minutes, or as much astwo hours, or more Human tissues of interest include, withoutlimitation, lung (including trachea and airways), liver, pancreas,testis, and the like. Physiological, biochemical, genomic or otherstudies can be carried out on the inhibitor-treated tissue to identifynovel therapeutic target molecules that are important in thepathophysiology of a disease. For example, isolated tissue from humanswithout CF can be exposed to inhibitor sufficient to induce the CFphenotype and such studies can be carried out to identify noveltherapeutic target molecules that are important in the pathophysiologyof CF.

EXAMPLES

[0112] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

[0113] The following methods and materials are used in the examplesbelow.

[0114] Cell Lines, Mice and Compounds

[0115] Fischer rat thyroid (FRT) cells coexpressing human wildtype CFTRand the halide indicator YFP-H148Q were generated as describedpreviously (Galietta et al. 2001 J. Biol. Chem. 276:19723-19728). Cellswere plated in 96-well black-walled microplates (Corning Costar) at adensity of 20,000 cells per well in Coon's modified F12 mediumsupplemented with 5% fetal calf serum, 2 mM L-glutamine, 100 U/mlpenicillin, and 100 μg/ml streptomycin. Assays were done at 48 h afterplating at which time cells were just confluent (˜40,000 cells perwell).

[0116] Initial screening was done using a diverse collection of 50,000drug-like compounds from ChemBridge (San Diego, Calif.) obtained as 10mM stock solutions in DMSO and diluted to 100 mM in 96-well microplates.Structure-activity analysis was done on analogs purchased fromChemBridge and ChemDiv (San Diego, Calif.).

[0117] Wildtype and cystic fibrosis (ΔF508 homozygous mutant) mice werebred by the CF Animal Core facility at U.C.S.F. Animal protocols wereapproved by the U.C.S.F. Committee on Animal Research.

[0118] Synthesis of 2-thioxo-4-thiazolidinone Analogs

[0119] Synthesis of3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(referred to herein as CFTR_(inh)-172) (see FIG. 1C) and analogs withdifferent positions of the trifluoromethyl and carboxy substituents(see, e.g., FIG. 1D) was accomplished by Knoevenagel condensation of2-thioxo-3-[a-trifluoromethyl-4-phenyl]-4-thiazolidinone (a=2, 3 or 4)with b-carboxybenzaldehyde (b=2, 3 or 4) in the presence of piperidine.Precipitate were filtered, washed with ethanol, dried and recrystallized2-3 times from ethanol to give bright yellow crystals (70-85% yields).Structures were confirmed by ¹H-NMR. Purity was >99% as judged by thinlayer chromatography and HPLC.

[0120] Screening Procedures

[0121] Assays were done using a customized screening system (Beckman)consisting of a 3-meter robotic arm, CO₂ incubator, plate washer, liquidhandling workstation, bar code reader, delidding station, and twoFluoStar fluorescence plate readers (BMG Labtechnologies, Offenburg,Germany), each equipped with two syringe pumps and HQ500/20X (500±10 nm)excitation and HQ535/30M (535±15 nm) emission filters (Chroma). Therobotic system was integrated using SAMI version 3.3 software (Beckman)modified for two plate readers. Custom software was written in VBA(Visual Basic for Applications) to compute baseline-subtracted,normalized fluorescence slopes (giving halide influx rates) from storeddata files.

[0122] The assay was set-up by loading the incubator (37° C., 90%humidity, 5% CO₂) with 40-60 96-well plates containing the FRT cells,and loading a carousel with 96-well plates containing test compounds anddisposable plastic pipette tips. To initiate the assay, each well of a96-well plate was washed 3 times in PBS (300 μl/wash), leaving 50 μlPBS. Ten μl of a CFTR-activating cocktail (5 μM forskolin, 100 μM IBMX,25 μM apigenin in PBS) was added, and after 5 min one test compound (0.5μl of 1 mM DMSO solution) was added to each well to give 10 μM finalconcentration. After 10 min, 96-well plates were transferred to a platereader for fluorescence assay. Each well was assayed individually forCFTR-mediated I⁻ transport by recording fluorescence continuously (200ms per point) for 2 s (baseline) and then for 12 s after rapid (<0.5 s)addition of 160 μL of isosmolar PBS in which 137 mM Cl⁻ was replaced byI⁻.

[0123] Assays of Intracellular [CAMP] and Toxicity

[0124] [cAMP] and phosphatase assays were done as reported previously(Galietta et al. 2001 J. Biol. Chem. 276:19723-19728). Cell toxicity wasdone by the dihydrorhodamine method at 24 hours after cell incubationwith 0-1000 μM inhibitor. Animal toxicity was assessed by measurement ofserum chemistries and hematology (U.C.S.F. Clinical Laboratory) in miceat 5 days after daily intraperitoneal injections with 0-100 μg/kginhibitor.

[0125] MDR-1 Activity

[0126] MDR-1 activity was evaluated by measuring ³H-vincristineaccumulation in an immortalized human tracheal cell line, 9HTEo-/Dx, inwhich the endogenous expression of MDR-1 was upregulated by selection inincreasing concentrations of doxorubicin (Rasola et al. 1994 J. Biol.Chem. 269:1432-1436). Cells were seeded in 24-well microplates (200,000cells/well). After 48 hours, cells were washed with a solutioncontaining (in mM): 130 NaCl, 2 KCl, 1 KH₂PO₄, 2 CaCl₂, 2 MgCl₂, 10Na-Hepes (pH 7.3) and 10 glucose, and incubated for 1 hour at 37° C.with 200 μl of the same solution containing ³H-vincristine (0.7 μM; 1μCi/ml). Cells were then washed three times with ice-cold solution andlysed in 0.25 M NaOH. Vincristine content was determined byscintillation counting.

[0127] Short-Circuit Current Tests

[0128] Snapwell inserts containing CFTR-expressing FRT cells or humanbronchial epithelial cells were mounted in an Ussing chamber system. ForFRT cells the hemichambers were filled with 5 ml of 75 mM NaCl and 75 mMNa gluconate (apical) and 150 mM NaCl (basolateral) (pH 7.3), and thebasolateral membrane was permeabilized with 250 μg/ml amphotericin B(Galietta et al. 2001 J Biol. Chem. 276:19723-19728). For bronchialepithelial cells and T84 cells, both hemichambers contained a Krebsbicarbonate solution. Hemichambers were continuously bubbled with air(FRT cells) or 5% CO₂ in air (bronchial and T84 cells) and maintained at37° C. Short-circuit current was recorded continuously using a DVC-1000voltage clamp (World Precision Instruments, Sarasota, Fla.) usingAg/AgCl electrodes and 1 M KCl agar bridges.

[0129] Patch-Clamp Analysis of Cl⁻ Channel Activity

[0130] Membrane current was measured in a whole-cell configuration. Forrecordings of Cl⁻ channels, the extracellular (bath) solution contained(in mM): 150 NaCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, 10 mannitol, 10 TES (pH7.4), and the intracellular (pipette) solution contained: 120 CsCl, 1MgCl₂, 10 TEA-Cl, 0.5 EGTA, 1 Mg-ATP, 10 Hepes (pH 7.3). CFTR wasactivated by forskolin (5 μM) in the extracellular solution. Thetime-course of membrane conductance was monitored in response toalternating voltage pulses of −100 and +80 mV. At defined times theprotocol was interrupted to generate current-voltage relationships(voltage pulses from −100 to +100 mV in 20 mV increments).Volume-sensitive Cl⁻ channels were activated by a hypotonic solution(extracellular NaCl decreased to 120 NaCl; 250 mosM/kg).Calcium-sensitive Cl⁻ channels were activated in human bronchialepithelial cells by addition of 100 μM UTP to the extracellularsolution.

[0131] Patch-Clamp Analysis of ATP-Sensitive K⁺ Channels

[0132] Membrane potential was recorded in the pancreatic, cell lineINS-1 in which the extracellular (bath) solution contained (in mM): 130NaCl, 2 KCl, 1 KH₂PO₄, 2 CaCl₂, 2 MgCl₂, 10 Na-Hepes (pH 7.3) and 10glucose. The pipette contained (in mM): 140 KCl, 1 CaCl₂, 2 mM MgCl₂, 10EGTA, 0.5 MgATP, 10 K-Hepes (pH 7.3). After achieving the whole-cellconfiguration, the amplifier was switched to current-clamp mode.

[0133] Intestinal Fluid Secretion and Short-circuit Current

[0134] In the first of 3 assays, fluid accumulation in ileal loops wasmeasured (Oi et al. 2002 Proc. Natl. Acad. Sci. USA 99:3042-3046;Gorbach et al. 1971 J. Clin. Invest. 50:881-889). Mice (age 8-10 weeks,body weight 25-35 g) in a CD1 genetic background (or ΔF508 homozygousmice) were starved for 24 hrs and anaesthetized with intraperitonealketamine (40 mg/kg) and xylazine (8 mg/kg). Body temperature wasmaintained during surgery at 36-38° C. using a heating pad. A smallabdominal incision was made to expose the small intestine and closedileal loops (length 20-30 mm) proximal to the cecum were isolated bysutures. Loops were injected with 100 μl of PBS alone or PBS containingcholera toxin (1 μg). In some experiments the inhibitor (150 μg/kg) wasadministered by intraperitoneal injection. The abdominal incision wasclosed with suture and mice were allowed to recover from anesthesia. At6 hours the mice were anesthestized, intestinal loops were exteriorized,and loop length and weight were measured after removal of mesentery andconnective tissue.

[0135] In the sealed adult mouse model of secretory diarrhea mice weregavaged with cholera toxin (10 μg) in 0.1 ml of 7% bicarbonate buffer(or buffer alone) using a orogastric feeding needle (Richardson et al.1986 Infect. Immun. 54:522-528; Gabriel et al. 1999 Am J. Physiol.276:G58-G63). Four experimental groups were: control (buffer alone),cholera-treated, cholera-treated+inhibitor (150 μg/kg intraperitoneal 2min before gavage), and inhibitor alone. After six hours mice wereeuthanized and the small intestine (from pylorus to cecum) wasexteriorized and stripped of associated mesenteric and connectivetissues. The intestine was weighed, then opened longitudinally to removelumenal fluid (by blotting), and weighed again. Fluid accumulation wascomputed from the ratio in intestinal weight before and after lumenalfluid removal. For measurement of short-circuit current, strips of ratcolon were isolated, stripped of muscle layers by blunt dissection,mounted in Ussing chambers (area 0.7 cm²), and bathed in oxygenatedbicarbonate Ringers solution containing 10 μM indomethacin.Short-circuit current was measured after inhibition of Na⁺ current byamiloride (10 μM), followed by stimulation by forskolin (20 μM) andsubsequent inhibitor addition.

Example 1

[0136] Screening of CFTR Inhibitors

[0137] The primary screening technique used to identify the compounds ofthe invention was designed to identify inhibitors of CFTR Cl⁻conductance by direct CFTR-inhibitor interaction. CFTR waspre-stimulated in CFTR-expressing FRT cells by an activating cocktailcontaining forskolin, IBMX and apigenin, as shown schematically in FIG.1A. The activation of CFTR by multiple mechanisms (cAMP elevation,phosphodiesterase inhibition, and direct CFTR binding) allowedidentification of inhibitors that blocked the CFTR Cl⁻ transportingpathway directly rather than more proximal step(s) in a signalingpathway. The FRT cells co-expressed a yellow fluorescent protein-basedCl⁻/I⁻ sensor that provided a quantitative fluorescence read-out ofinhibition potency (See, e.g., Jayaraman et al., 2000, J. Biol. Chem.275:6047-6050; Galietta et al., 2001, Am. J. Physiol. 281:C1734-C1742.).After CFTR pre-stimulation and compound addition, cells were subjectedto an inwardly-directed I⁻ gradient to drive I⁻ influx and producedecreasing fluorescence. Each assay consisted of recording baselinefluorescence for 2 seconds, followed by 12 seconds of continuousrecording of fluorescence after rapid addition of the I⁻ containingsolution. Compounds were tested separately at 10 μM concentration in a96-well format utilizing a fully-automated high-throughput screeningapparatus (see Example 2 below).

[0138]FIG. 1B graphically illustrates representative curves, as relativeYFP fluorescence versus time, from the primary screen of 50,000compounds using the assay of FIG. 1A. As quantified from the slope ofthe decreasing fluorescence after I⁻ addition, 49,993 compounds had nosignificant effect on the kinetics of I⁻ influx (<10% decrease inslope). Seven compounds produced a small decrease in negative slope(10-52%), nearly all of which had a similar core structure consisting ofa 2-thioxo-4-thiazolidinone heterocycle with substituted phenylmethyleneand phenyl moieties (FIG. 1C). More than 250 analogs havingthiazolidinone core structure were subsequently screened to identify themost potent CFTR inhibitors.

[0139]FIG. 1D shows the most effective thiazolidinone CFTR inhibitorsidentified in the screening were3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(referred to herein as CFTR_(inh)-172), along with five analogs havingsignificant inhibitory potencies. Thus the following compounds wereidentified as CFTR inhibitors: 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(CFTR_(inh)-172);3-[(3-trifluoromethyl)phenyl]-5-[(4-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone(CFTR_(inh)-020);3-[(3-trifluoromethyl)phenyl]-5-[(4-oxycarboxyphenyl)methylene]-2-thioxo-4-thiazolidinone(CFTR_(inh)-029);3-[(3-trifluoromethyl)phenyl]-5-[(3,4-dihydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone(CFTR_(inh)-185),3-[(3-trifluoromethyl)phenyl]-5-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone(CFTR_(inh)-214) and3-[(3-trifluoromethyl)phenyl]-5-[(3-bromo-4-hydroxy-5-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone(CFTR_(inh)-236). The most effective CFTR inhibitors included one ormore electronegative groups such as a 3-trifluoromethyl group on ring 1,and electronegative or polar substituents on ring 2 as discussed above.CFTR_(inh)-172 was selected for further analysis. The relative potencieswere: 0.2 (CFTR_(inh)-020), 0.3 (CFTR_(inh)-029), 1.0 (CFTR_(inh)-172),0.2 (CFTR_(inh)-185), 0.1 (CFTR_(inh)-214), and 0.1 (CFTR_(inh)-236).

[0140] To examine the effect of ring position of the trifluoromethyl andcarboxyl substituents, 8 analogs of CFTR_(inh)-172 were synthesized inwhich the substituents were moved to each unique position on rings 1(trifluoromethyl) and 2 (carboxy). Compared to CFTR_(inh)-172 (potency1.0), the relative inhibitory potencies of the3-[(a-trifluoromethyl)phenyl]-5-[(b-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone analogs were: 0.69 (a=2, b=2), 0.70(2, 3), 0.66 (2, 4), 0.74 (3, 2), 0.90 (3, 3), 0.67 (4, 2), 0.64 (4, 3)and 0.56 (4, 4).

Example 2

[0141] Characterization of CFTR_(inh)-172

[0142] The level of CFTR inhibition for specific dosages of the subjectthiazolidinone compounds was determined using the fluorescence assayshown in FIG. 1A and described above. FIG. 2A shows dose-inhibition datafor CFTR_(inh)-172 as relative YFP fluorescence versus time. SignificantCFTR inhibition was seen at 0.3-0.6 μM concentrations of thisthiazolidinone compound. FIG. 2B shows that inhibition by CFTR_(inh)-172(shown graphically as relative transport rate versus time after additionor washout) was complete in ˜10 min (t_(1/2) 4 min) and was reversedafter washout with t_(1/2) ˜5 min (inset). The relative transport ratesillustrated in FIG. 2C show that CFTR_(inh)-172 effectively inhibitedCFTR activation by multiple types of agonists that were not included inthe activating cocktail used for initial screening. These agonistsincluded genistein, CPT-cAMP, CPX, 8-MPO and the potent benzoflavoneCFTR activator UCCF-029 (2-(4-pyridinium)benzo[h]4H-chromen-4-onebisulfate) and the benzimidazolone CFTR activator UCCF-853 (seeGalietta, et al., 2001, J. Biol. Chem. 276:19723-19728).

[0143] Electrophysiology experiments were also carried out to establishthe inhibitory potency and specificity of CFTR_(inh)-172. FIG. 3A showsthe rapid, dose-dependent inhibition of short-circuit current inCFTR-expressing FRT cells with CFTR_(inh)-172 added to the solutionbathing the apical cell surface. FIG. 3B shows the averagedose-inhibition relationships of CFTR_(inh)-172 (K_(d)˜300 nM, Hillcoefficient ˜1) and glibenclamide (K_(d)˜200 μM) tested under identicalconditions.

[0144] Similar inhibitory potencies for this thiazolidinone were foundin cells that natively express wildtype CFTR, including T84 cells andprimary cultures of human bronchial epithelial cells, as well as intransfected FRT cells expressing G551D-CFTR and ΔF508-CFTR (after lowtemperature correction). For studies in bronchial cells, the Na⁺ channelwas blocked with amiloride so that baseline current is largelyCFTR-dependent. After maximal CFTR activation by a CPT-cAMP, applicationof CFTR_(inh)-172 from the apical side inhibited short-circuit currentstrongly (FIG. 3C, left). CFTR_(inh)-172 also inhibited short-circuitcurrent when added from the basolateral side (FIG. 3C, right).

[0145] Whole-cell membrane currents were measured in CFTR-expressing FRTcells as shown in FIG. 3D. Stimulation by 5 μM forskolin produced amembrane current of 381±47 pA/pF (n=4) at +100 mV (total membranecapacitance 21±3 μF). The current-voltage relationship was linear asexpected for a pure CFTR current (FIG. 3F). Extracellular perfusion with2 μM CFTR_(inh)-172 produced a rapid reduction in current at allmembrane potentials, suggesting voltage-independent CFTR inhibition. Thelack of voltage-dependence of channel block was confirmed using a lowerconcentration of CFTR_(inh)-172 (0.2 μM) to obtain ˜50 % inhibition(FIG. 3F).

[0146] The specificity of CFTR_(inh)-172 for inhibition of CFTR was alsoexamined. Two non-CFTR Cl⁻ channes were studied. CFTR_(inh)-172 at 5 μMconcentration did not inhibit Ca²⁺ activated Cl⁻ secretion produced byaddition of UTP (100 μM) to the apical bathing solution in polarizedhuman bronchial epithelial cells (FIG. 4A). Maximal UTP-dependentshort-circuit currents were 9.9±0.5 μA/cm² and 10.0±0.2 μA/cm² in theabsence and presence of CFTR_(inh)-172, respectively (SE, n=4).CFTR_(inh)-172 at 5 μM also did not block volume-activated Cl⁻ currentselicited in FRT cells by extracellular perfusion with a 250 mosM/kghypotonic solution (FIG. 4B).

[0147] The activity of a CFTR homolog, the ATP-binding cassettetransporter MDR-1 (multi-drug resistance protein-1), was measured in9HTEo-/Dx which overexpress MDR-1 (Rasola et al. 1994 J. Biol. Chem.269:1432-1436). Vincristine accumulation, which is inversely related toactive drug extrusion by MDR-1, was strongly increased by the MDR-1inhibitor verapamil (100 μM) (FIG. 4C). CFTR_(inh)-172 (5 μM) did notaffect vincristine accumulation and thus did not inhibit MDR-1.

[0148] Another homolog of CFTR is the sulphonylurea receptor (SUR) whichregulates the activity of ATP-sensitive K⁺ channels (K-ATP channel)(Aguilar-Bryan and Bryan 1999 Endocr. Rev. 20:101-135). SUR1 isexpressed in pancreatic β-cells where it controls membrane potential andinsulin release. Sulphonylureas, like glibenclamide, cause insulinrelease (and a hypoglycemic response) by blocking K-ATP channels andmembrane depolarization. To determine whether CFTR_(inh)-172 also blocksK-ATP channels, membrane potential in a rat pancreatic β cell line,INS-1, was measured (FIG. 4D, FIG. 4E). In contrast to large membranedepolarization caused by glibenclamide, CFTR_(inh)-172 (2 and 5 μM) didnot depolarize membrane potential. CFTR_(inh)-172 at 5 μM caused a smallhyperpolarization that was much less than that caused by the K-ATPchannel activator diazoxide (100 μM). Additional studies indicated thatCFTR_(inh)-172 at 5 μM did not block a water channel (AQP1), ureatransporter (UT-B), Na⁺/H⁺ exchanger (NEE3) and Cl⁻/HCO₃ ⁻ exchanger(AEI).

[0149] Further analysis showed that 5 μM CFTR_(inh)-172 did not affectcellular cAMP production or phosphatase activity. In FRT cells, basalcAMP content was 225±22 fmol/well, which increased at 30 min afterstimulation by 20 μM forskolin to 1290±190 fmol/well (no inhibitor) and1140±50 (+CFTR_(inh)-172) (n=3). As judged using the dihydrorhodamineassay, CFTR_(inh)-172 was non-toxic to FRT cells after 24 hours atconcentrations up to 100 μM. In mice, intraperitoneal injection of 1000μg/kg CFTR_(inh)-172 daily for 7 days did not cause overt toxicity. Foodand water intake were not diminished, and serum electrolyteconcentrations, glucose, liver function indices, serum creatinine,amylase and hematocrit were not changed. In addition, a single verylarge systemic dose of CFTR_(inh)-172 (10 mg/kg) did not cause overttoxicity.

Example 3

[0150] In Vivo Efficacy

[0151] The efficacy of CFTR_(inh)-172 was tested in vivo using twoassays of cholera toxin-induced intestinal fluid secretion, and inisolated intestine by short-circuit analysis. In the first assay, aseries of closed loops of small intestine were created in vivo and thelumens of alternate loops were injected with small volumes of saline orsaline containing cholera toxin. Luminal fluid accumulation wasdetermined after 6 hours. As seen visually in FIG. 5A, there was markedfluid accumulation and distention in cholera toxin-treated loops,whereas adjacent control (saline) loops remained empty. A singleadministration of CFTR_(inh)-172 (150 μg/kg intraperitoneal) prior tocholera toxin infusion effectively prevented fluid accumulation in thetoxin-treated intestinal loops.

[0152] Data from a series of these experiments is summarized graphicallyin FIG. 5B. CFTR_(inh)-172 significantly reduced fluid secretion to thatin saline control loops where an inactive thiazolidinone analog did notinhibit fluid secretion. As suggested from previous data (Gabriel et al.1994 Science 266:107-109), cholera toxin-treated loops of intestine fromhomozygous ΔF508-CFTR mice also remained empty, indicating theinvolvement of CFTR in intestinal fluid secretion. In the second assay,intestinal fluid secretion was induced by oral administration of choleratoxin (10 μg) and CFTR_(inh)-172 was administered systemically. Aftersix hours there was marked accumulation of fluid as measured by weighingthe entire small intestine. CFTR_(inh)-172 administration remarkablyreduced intestinal fluid accumulation as seen visually and quantified bythe ratio of intestinal weight before vs. after luminal fluid removal(FIG. 5C).

[0153]FIG. 5D shows CFTR_(inh)-172 inhibition of short-circuit currentacross intact rat colonic mucosa. After inhibition of Na⁺ current byamiloride, forskolin produced a prompt increase in short-circuitcurrent. CFTR_(inh)-172 added to the mucosal solution inhibitedshort-circuit current with greater efficacy than when added to theserosal solution, which may be related to impaired access to colonicepithelial cells through the residual submucosal tissue. Addition of 5μM CFTR_(inh)-172 to the mucosal solution alone reduced short-circuitcurrent by >80%. These results provide electrophysiological evidence forCFTR Cl⁻ channel inhibition by CFTR_(inh)-172 in intestine.

[0154] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of treating a subject having a condition associated withaberrant ion transport by cystic fibrosis transmembrane conductanceregulator (CFTR) in a subject, the method comprising: administering tothe subject an efficacious amount of a thiazolidinone compound; whereinCFTR ion transport is inhibited and the condition is treated.
 2. Themethod of claim 1, wherein the aberrantly increased CFTR ion transportis associated with diarrhea.
 3. The, method of claim 2, wherein thediarrhea is secretory diarrhea.
 4. The method of claim 1, wherein saidthiazolidinone compound comprises a3-aryl-5-arylmethylene-2-thioxo-4-thiazolidinone.
 5. The method of claim1, wherein said thiazolidinone compound is selected from the groupconsisting of:3-[(3-trifluoromethyl)phenyl]-5-[(4-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(4-oxycarboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(3,4-dihydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone;and3-[(3-trifluoromethyl)phenyl]-5-[(3-bromo-4-hydroxy-5-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone.6. The method of claim 1, wherein said thiazolidinone compound comprisesthe formula:

wherein X₁, X₂ and X₃ each individually are hydrogen, any organic group,any halo group, a nitro group, an azo group, a hydroxyl group or a thiogroup, Y₁, Y₂ and Y₃ each individually are hydrogen, any organic group,any halo group, a nitro group, an azo group, a hydroxyl group or a thiogroup, A₁ and A₂ each individually are oxygen or sulfur, A₃ is sulfur orselenium, and A₄ comprises one or more carbons or heteroatoms and may bepresent or absent.
 7. The method of claim 1, wherein said thiazolidinonecompound comprises the formula:

wherein X is hydrogen, any organic group, any halo group, a nitro group,an azo group, a hydroxyl group or a thio group, Y₁, Y₂ and Y₃ eachindividually are hydrogen, any organic group, any halo group, a nitrogroup, an azo group, a hydroxyl group or a thio group, and A₁ and A₂each independently are oxygen or sulfur.
 8. The method of claim 7,wherein X is an electronegative group.
 9. The method of claim 8, whereinX is selected from the group consisting of a perfluoroalkyl group and afluoro group.
 10. The method of claim 9, wherein Y1 is selected from thegroup consisting of alkyl, hydroxyl, carboxyl, nitro, carbonate,carbamate, alkoxy, alkylcarbonyl, and halo groups.
 11. The method ofclaim 8, wherein X is a 3-trifluoromethyl group.
 12. The method of claim7, wherein Y1 is a hydroxyl group.
 13. The method of claim 12, whereinY2 is a hydroxyl group.
 14. The method of claim 12, wherein Y2 is abromo group.
 15. The method of claim 12, wherein Y3 is a nitro group.16. The method of claim 1, wherein said thiazolidinone compoundcomprises the formula:

wherein X is hydrogen or any organic group, Y1, Y2 and Y3 eachindividually are hydrogen or any organic group.
 17. The method of claim1, wherein said thiazolidinone compound comprises the formula:

wherein X is any electronegative group or electron withdrawing group,and Y₁, Y₂ and Y₃ each individually are a hydrogen, alkyl, hydroxyl,carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, or halogroup.
 18. The method of claim 17, wherein X is a trifluoromethyl group.19. The method of claim 18, wherein X is a 3-trifluoromethyl group. 20.The method of claim 1, wherein said thiazolidinone compound comprises aformula selected from the group consisting of:


21. A method for inhibiting cystic fibrosis transmembrane conductanceregulator protein in cells of a subject, comprising contacting saidcells with an efficacious amount of a thiazolidinone compound.
 22. Themethod of claim 21, wherein said thiazolidinone compound comprises a3-aryl-5-arylmethylene-2-thioxo-4-thiazolidinone.
 23. The method ofclaim 21, wherein said thiazolidinone compound is selected from thegroup consisting of:3-[(3-trifluoromethyl)phenyl]-5-[(4-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(4-oxycarboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(3,4-dihydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone;and3-[(3-trifluoromethyl)phenyl]-5-[(3-bromo-4-hydroxy-5-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone.24. The method of claim 21, wherein said thiazolidinone compoundcomprises the formula:

wherein X₁, X₂ and X₃ each individually are hydrogen, any organic group,any halo group, a nitro group, an azo group, a hydroxyl group or a thiogroup, Y₁, Y₂ and Y₃ each individually are hydrogen, any organic group,any halo group, a nitro group, an azo group, a hydroxyl group or a thiogroup, A₁ and A₂ each individually are oxygen or sulfur, A₃ is sulfur orselenium, and A₄ comprises one or more carbons or heteroatoms and may bepresent or absent.
 25. The method of claim 21, wherein saidthiazolidinone compound comprises the formula:

wherein X is hydrogen, any organic group, any halo group, a nitro group,an azo group, a hydroxyl group or a thio group, Y₁, Y₂ and Y₃ eachindividually are hydrogen, any organic group, any halo group, a nitrogroup, an azo group, a hydroxyl group or a thio group, and A₁ and A₂each independently are oxygen or sulfur.
 26. The method of claim 25,wherein X is an electronegative group.
 27. The method of claim 26,wherein X is selected from the group consisting of a perfluoroalkylgroup and a fluoro group.
 28. The method of claim 25, wherein Y1 isselected from the group consisting of alkyl, hydroxyl, carboxyl, nitro,carbonate, carbamate, alkoxy, alkylcarbonyl, and halo groups.
 29. Themethod of claim 26, wherein X is a 3-trifluoromethyl group.
 30. Themethod of claim 25, wherein Y1 is a hydroxyl group.
 31. The method ofclaim 30, wherein Y2 is a hydroxyl group.
 32. The method of claim 30wherein Y2 is a bromo group.
 33. The method of claim 30, wherein Y3 is anitro group.
 34. The method of claim 21, wherein said thiazolidinonecompound comprises the formula:

wherein X is hydrogen or any organic group, Y1, Y2 and Y3 eachindividually are hydrogen or any organic group.
 35. The method of claim21, wherein said thiazolidinone compound comprises the formula:

wherein X is any electronegative group or electron withdrawing group,and Y₁, Y₂ and Y₃ each individually are a hydrogen, alkyl, hydroxyl,carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, or halogroup.
 36. The method of claim 35, wherein X is a trifluoromethyl group.37. The method of claim 36, wherein X is a 3-trifluoromethyl group. 38.The method of claim 21, wherein said thiazolidinone compound comprises aformula selected from the group consisting of:


39. The method of claim 21, wherein said contacting said cells iscarried out in vivo in a subject.
 40. The method of claim 21, whereinsaid contacting said cells comprises ingesting, by said subject, saidthiazolidinone compound.
 41. The method of claim 40, wherein saidingesting further comprises ingesting a pharmaceutically acceptablecarrier together with said thiazolidinone compound.
 42. A pharmaceuticalcomposition comprising a thiazolidinone compound together with at leastone of a pharmaceutically acceptable carrier, a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable excipient and apharmaceutically acceptable adjuvant, said thiazolidinone compoundselected from the group consisting of:3-[(3-trifluoromethyl)phenyl]-5-[(4-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(oxycarboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(3,4-dihydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone;3-[(3-trifluoromethyl)phenyl]-5-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-thioxo-4-thiazolidinone;and3-[(3-trifluoromethyl)phenyl]-5-[(3-bromo-4-hydroxy-5-nitrophenyl)methylene]-2-thioxo-4-thiazolidinone.43. The composition of claim 42, wherein said composition does notcontain dimethyl sulfoxide.
 44. A pharmaceutical composition comprisinga thiazolidinone compound together with at least one of apharmaceutically acceptable carrier, a pharmaceutically acceptablediluent, a pharmaceutically acceptable excipient and a pharmaceuticallyacceptable adjuvant, said thiazolidinone compound having a formula:

wherein X₁, X₂ and X₃ each individually are hydrogen, any organic group,any halo group, a nitro group, an azo group, a hydroxyl group or a thiogroup, Y₁, Y₂ and Y₃ each individually are hydrogen, any organic group,any halo group, a nitro group, an azo group, a hydroxyl group or a thiogroup, A₁ and A₂ each individually are oxygen or sulfur, A₃ is sulfur orselenium, and A₄ comprises one or more carbons or heteroatoms and may bepresent or absent.
 45. The composition of claim 44, wherein saidthiazolidinone compound comprises the formula:

wherein X is hydrogen, any organic group, any halo group, a nitro group,an azo group, a hydroxyl group or a thio group, Y₁, Y₂ and Y₃ eachindividually are hydrogen, any organic group, any halo group, a nitrogroup, an azo group, a hydroxyl group or a thio group, and A₁ and A₂each independently are oxygen or sulfur.
 46. The composition of claim45, wherein X is an electronegative group.
 47. The composition of claim46, wherein X is selected from the group consisting of a perfluoroalkylgroup and a fluoro group.
 48. The composition of claim 45, wherein Y1 isselected from the group consisting of alkyl, hydroxyl, carboxyl, nitro,carbonate, carbamate, alkoxy, alkylcarbonyl, and halo groups.
 49. Thecomposition of claim 45, wherein X is a 3-trifluoromethyl group.
 50. Thecomposition of claim 45, wherein Y1 is a hydroxyl group.
 51. Thecomposition of claim 50, wherein Y2 is a hydroxyl group.
 52. Thecomposition of claim 50, wherein Y2 is a bromo group.
 53. The method ofclaim 52, wherein Y3 is a nitro group.
 54. The composition of claim 44,wherein said thiazolidinone compound comprises the formula:

wherein X is hydrogen or any organic group; and Y1, Y2, and Y3 eachindividually are hydrogen or any organic group.
 55. The composition ofclaim 54, wherein said thiazolidinone compound comprises the formula:

wherein X is any electronegative group or electron withdrawing group,and Y₁, Y₂ and Y₃ each individually are a hydrogen, alkyl, hydroxyl,carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, or halogroup.
 56. The composition of claim 55, wherein X is a trifluoromethylgroup.
 57. The composition of claim 55, wherein X is a 3-trifluoromethylgroup.
 58. The composition of claim 44, wherein said composition doesnot contain dimethyl sulfoxide.
 59. A non-human animal having a cysticfibrosis transmembrane conductance regulator (CFTR) deficiency, whereinthe deficiency is produced by administration of a thiazolidinonecompound to the animal in an amount effective to inhibit CFTR iontransport.
 60. The non-human animal of claim 59, wherein the animal is amammal.
 61. The non-human animal of claim 60, wherein the mammal is anon-human primate, rodent, ungulate, or avian.
 62. The non-human animalof claim 59, wherein the animal has a phenotype similar to cysticfibrosis.